Various methods have been developed for the amplification of nucleic acids, including Polymerase Chain Reaction (PCR), Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification. Each of these methods involves a series of complex and sensitive steps and an accurate, qualitative and/or quantitative assay requires that variability at each step is carefully controlled. It is also critical that the control standards are precisely calibrated and they can withstand the rigors of the assay procedures.
Qualitative detection of a nucleic acid in a biological sample is crucial, e.g. for recognizing an infection of an individual, and therefore false-negative and false-positive results should be minimized. Thus, a qualitative internal control nucleic acid is added to the detection mix. Moreover, quantifying viral nucleic acid sequences in a biological sample is an important tool for assessing a patient's viral load, i.e., the measure of the total quantity of viral particles within a given patient at one point in time. In chronic infections, viral load is a function of a highly dynamic equilibrium of viral replication and immune-mediated host clearance. Viral load can be used to assess the degree of viral replication at the time of diagnosis which provides an assessment of the patient's progression and prognosis. It can also be used to monitor the effect of antiviral medications early in the disease course and quickly assess the effects of changing antiviral medications.
Numerous nucleic acids relevant for clinical diagnostics are ribonucleic acids, e.g. the nucleic acids from RNA viruses such as, for example, Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), West Nile Virus (WNV), Human Papilloma Virus (HPV), Japanese Encephalitis Virus (JEV), St. Louis Encephalitis Virus (SLEV), Influenza Virus, Norovirus, and others. Therefore, it can be advantageous to use an internal control nucleic acid consisting of RNA in order to reflect the properties of the target nucleic acids in the sample. Because RNA is more prone to degradation than DNA due to its inherent sensitivity to influences such as alkaline pH, ribonucleases, etc., internal control nucleic acids made of RNA are commonly provided as armored particles. Armored RNA® (aRNA) technologies stabilize and protect nucleic acids from nuclease degradation by packaging them in a protective bacteriophage protein coat. (Armored RNA is developed by Ambion, Inc. and Cenetron Diagnostics LLC, both of Austin, Tex., and covered under U.S. Pat. Nos. 5,677,124, 5,919,625 and 5,939,262, the disclosures of which are incorporated herein by reference in their entireties.) These aRNA constructs are fully processed within the assay, they are thermostable at ambient temperatures, and they are relatively RNase resistant. However, at high temperatures, aRNA constructs rapidly degrade, resulting in assay loss. In order to extend the shelf life of products including these constructs, the products are shipped and stored at 4° C., which is costly. Therefore, there is a need for a heat resistant, ribonuclease-resistant RNA standard for use in qualitative and/or quantitative nucleic acid detection methods.